Evaluating the potential of non‐immunosuppressive cyclosporin analogs for targeting Toxoplasma gondii cyclophilin: Insights from structural studies

Abstract Toxoplasmosis persists as a prevalent disease, facing challenges from parasite resistance and treatment side effects. Consequently, identifying new drugs by exploring novel protein targets is essential for effective intervention. Cyclosporin A (CsA) possesses antiparasitic activity against Toxoplasma gondii, with cyclophilins identified as possible targets. However, CsA immunosuppressive nature hinders its use as an antitoxoplasmosis agent. Here, we evaluate the potential of three CsA derivatives devoid of immunosuppressive activity, namely, NIM811, Alisporivir, and dihydrocyclosporin A to target a previously characterized cyclophilin from Toxoplasma gondii (TgCyp23). We determined the X‐ray crystal structures of TgCyp23 in complex with the three analogs and elucidated their binding and inhibitory properties. The high resolution of the structures revealed the precise positioning of ligands within the TgCyp23 binding site and the details of protein–ligand interactions. A comparison with the established ternary structure involving calcineurin indicates that substitutions at position 4 in CsA derivatives prevent calcineurin binding. This finding provides a molecular explanation for why CsA analogs can target Toxoplasma cyclophilins without compromising the human immune response.


Table of Contents Table
. Binding energy and predicted binding affinity values from in silico docking of best-scoring compounds.Table S2.Data collection and refinement statistics for all TgCyp23 structures presented in this study.Table S3.Polar contacts between TgCyp23 and CsA/dhCsA/NIM811/Alisporivir. Figure S1.Interaction patterns of CsA analogs with TgCyp23. Figure S2. 1 H-15 N HSQC NMR spectra of 15 N-labelled TgCyp23 in the absence and presence of a two-fold molar excess of CsA. Figure S3.Chemical shift perturbation difference between the TgCyp23:CsA complex and TgCyp23 in complex with the CsA-analogs.

Figure S1 .
Figure S1.Interaction patterns of CsA analogs with TgCyp23.(A) TgCyp23:CsA complex, CsA in orange sticks.(B) TgCyp23:dhCsA complex, dhCsA in pink sticks.(C) TgCyp23:NIM811 complex, NIM811 in purple sticks.(D) TgCyp23:Alisporivir complex, Ali in cyan sticks.The protein is displayed as a grey cartoon, and relevant residues implicated in the interaction are labeled and shown as sticks.Black dashed lines indicate polar interactions, and the red dashed line represents Van der Waals forces.

Figure S2. 1 H
Figure S2. 1 H-15 N HSQC NMR spectra of 15 N-labelled TgCyp23 in the absence (black) and presence of a two-fold molar excess of CsA (red).Enlarged views of selected spectral regions that show significant chemical shift perturbation upon CsA binding are displayed.Significantly perturbed protein residues are also labelled with single-letter amino acid code.

Figure S3 .
Figure S3.Chemical shift perturbation difference between the TgCyp23:CsA complex and TgCyp23 in complex with the CsA-analogs.Alisporivir (upper panel), NIM811 (middle panel) and dhCsA (lower panel).Secondary structure elements are displayed.

Figure S4 .
Figure S4.Interaction of TgCyp23 with CsA analogs studied by ITC.(A-C) Representative thermograms (top panels) and the derived binding isotherms (bottom panels) of TgCyp23 in complex with dhCsA (A), NIM811 (B) and Alisporivir (C) recorded at 20 °C.(D-E) Blank titrations were performed by injecting dhCsA (D), NIM811 (E), and Alisporivir (F) into the buffer devoid of protein to correct for the heat of mixing and ligand dilution.This contribution to the observed heat of reaction was subtracted from the corresponding total heat.

Figure S5 .
Figure S5.Comparison of TgCyp23:CsA complex with Homo sapiens CypA:CsA complex (PDB: 1M63).(A) Alignment between CypA (Blue) and TgCyp23 (grey).Structures are displayed in cartoon view.Right panel shows a 60º rotated view.(B) Representation of the molecular surface of CypA (colored in blue) showing the CsA binding pocket (CsA depicted as orange capped sticks).(C) Representation of the molecular surface of TgCyp23 (colored in grey) showing the CsA binding pocket (CsA depicted as orange capped sticks).